Interpretive Summary: The global focus on the environment has necessitated the development of new processes to better utilize renewable materials to provide sustainable technologies and alleviate greenhouse gas emissions. One technology that has garnered increased attention is biofuels. Traditionally, biofuels have been understood to be any fuel that has its origins from natural precursors. These would include ethanol, biodiesel, and hydrogen among others. Within the past 10 years biodiesel has exploded onto the biofuels scene and has become the “biofuel of choice” for blending in many applications. Among its many benefits, sustainability and emissions reduction make biodiesel a friendlier alternative to petroleum-based diesel. Unfortunately, biodiesel production results in a large co-product stream that is high in glycerine which cuts into profit margins because of the high costs associated with glycerine removal. Therefore, in order to aid biodiesel producers efforts are underway to find new uses for glycerine in order to help it maintain value. One area that is currently being studied is the use of glycerine as a fermentation feedstock (food for microorganisms) for the production of new “environmentally benign” materials. Poly(hydroxyalkanotes) (PHA) are polyesters (plastics) that are made by a number of bacterial species. Many of these molecules have been shown to maintain properties that are analogous to polyethylene and polypropylene with the added advantage of biodegradability and sustainability. In the present report we document our successes in utilizing glycerine along with levulinic acid (an inexpensive by-product of the pulp and paper industry) as mixed feedstocks for the production of PHA polymers with diverse properties. By utilizing these low-cost feedstocks, we have demonstrated a new potential outlet for these materials while at the same time provided a more economical means to produce renewable bioplastics. Specifically, we show that by varying the glycerine:levulinic acid ratios in the growth media and the fermentation times, PHA polymers can be produced with tunable physical properties (different chemical compositions, sizes, etc.), which provide controlled strength, ductility and toughness for a variety of applications. This work has opened up a new avenue for glycerine and levulinic acid utilization in the formation of sustainable bioplastics with controlled properties thus benefiting both the biodiesel and the polymer (plastics) industries.

Technical Abstract:
Glycerine and levulinic acid were used alone and in combination for the fermentative synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/V) biopolymers. Shake-flask cultures of Pseudomonas oleovorans NRRL B-14682 containing different glycerine:levulinic acid ratios (1%, w/v total carbon source) resulted in polymers containing 3-hydroxybutyrate (3-HB) and 3-hydroxyvalerate (3-HV) with tunable monomer contents. Increased levulinic acid media content required longer culture times to achieve maximal cell productivities. Compositional analysis by 1H-NMR and GC/MS revealed that the use of glycerine alone resulted in poly(3-hydroxybutyrate) (PHB); however, when levulinic acid was added to the production media at initial concentrations less than or equal to 0.6 wt%, PHB/V copolymers were produced with 3-HV contents ranging from 37 to 97 mol%. A glycerine:levulinic acid substrate ratio of 0.2%:0.8% resulted in poly(3-hydroxyvalerate) (PHV) but levulinic acid in the absence of glycerine resulted in no bacterial growth and consequently no polymer production. Scale-up to 10-L bench-top fermentations provided sufficient polymers to assess the effects of monomer content and molecular weight on tensile properties. PHB, P(73%-3HB-co-27%-3HV), P(30%-3HB-co-70%-3HV) and PHV were produced at the 10-L scale with number average molecular weights (Mn) of 328,000, 511,000, 728,000 and 1,330,000 103 g/mol, respectively. Films were solution cast from CHCl3 and the tensile properties were measured at 3 days and at 2 months. Tensile strength remained relatively constant over time (+/- 1.3 MPa), but elongation at break and fracture energy (toughness) both decreased for each of the polymers resulting in increased Modulus (stress / strain) values. In each case the properties of the 3-HV-containing polymers were superior to PHB. The results of this work may provide an economic advantage for the production, property control and application of these sustainable biopolymers.